Elucidating spatio-temporal innate immune cell modulation of Mycobacterium tuberculosis stress response and replication heterogeneity

PROJECT SUMMARY/ABSTRACT Heterogeneity, including in Mycobacterium tuberculosis (Mtb) replication status and local environment, is a hallmark of Mtb infection and strongly impacts infection outcome and disease treatment. Spatial organization of Mtb lesions in the lung affect environmental signals that the bacteria receive, with consequent effects on Mtb stress responses and growth. For example, Mtb within the necrotic lesion core are extracellular, while Mtb in the lesion cuff reside within immune cells, resulting in exposure to different oxygen levels, pH and nitric oxide (NO) stress, among other signals. Mtb also exhibits shifts in metabolism over the course of infection, with lipid metabolism vital as infection progresses and lipid-rich foamy macrophages form. Importantly, environmental non-uniformity exists even within each lesion sublocation, likely driven by proximity to/residence in different immune cell types and their specialized antimicrobial programming. Consistent with this hypothesis, we observe a greater dependence on interstitial macrophages (IMs) in controlling Mtb growth in necrotic lesions versus alveolar macrophages (AMs). Different antimicrobial effectors in AMs and IMs may contribute to this difference in controlling Mtb growth. Notably, we observe an increase in extracellular lipid levels in the periphery of the necrotic lesion core upon macrophage depletion, corresponding with local expansion of the Mtb population. The neutral environment of the lesion core versus the slightly acidic macrophage phagosome compartment may represent a mechanism fueling this Mtb growth. Elucidating how macrophage subtypes act to restrict Mtb growth within lesions, and how Mtb stress responses and adaptation relates to macrophage subtypes in situ, at the single bacterium level, is thus critical for understanding Mtb infection biology. Using the C3HeB/FeJ murine infection model that recapitulates canonical human Mtb lesions, I will elucidate how AM/IM distribution in these lesions over time relate to changes in Mtb exposure to NO/hypoxia, acidic pH stress, and lipid utilization, which all have been shown to affect Mtb growth in vitro. To further probe the role of macrophages in containing Mtb growth within necrotic lesions, macrophage depletion will be performed after lesions have formed, with tests of inducible knockdown Mtb mutants in key lipid/pH regulators to provide mechanistic insight into how disruption of these pathways can alter the ability of Mtb to adapt for survival and growth in necrotic lesions. This proposal uses quantitative in situ mRNA transcript visualization techniques to assess how immune cells spatiotemporally drive key aspects of the local environment that impact Mtb growth in necrotic lesions, knowledge vital to the development of more effective anti-tubercular therapeutic regimens. Completion of the proposed studies will provide the necessary training in host-pathogen interactions to enable me to transition to starting my own independent laboratory at a research-intensive institution.